U.S. patent application number 12/197450 was filed with the patent office on 2009-08-13 for oligopeptides for use as taste modulators.
This patent application is currently assigned to Nutrinova Nutrition Specialties & Food Ingredients GmbH. Invention is credited to Michael Krohn, Holger Zinke.
Application Number | 20090202698 12/197450 |
Document ID | / |
Family ID | 39863089 |
Filed Date | 2009-08-13 |
United States Patent
Application |
20090202698 |
Kind Code |
A1 |
Krohn; Michael ; et
al. |
August 13, 2009 |
OLIGOPEPTIDES FOR USE AS TASTE MODULATORS
Abstract
The invention relates to the use of one or more oligopeptide/s,
preferably a tripeptide such as aladapcin, as a taste modulator
and/or sweetness enhancer for comestible compositions preferably
those containing at least one natural or artificial sweetener. The
comestible compositions include food, beverages, medicinal products
and cosmetics and contain preferably mono-, di- or oligosaccharides
as sweeteners. The invention further relates to said comestible
compositions containing an oligopeptide as taste modulator.
Inventors: |
Krohn; Michael; (Lorsch,
DE) ; Zinke; Holger; (Heppenheim, DE) |
Correspondence
Address: |
HAMMER & ASSOCIATES, P.C.
3125 SPRINGBANK LANE, SUITE G
CHARLOTTE
NC
28226
US
|
Assignee: |
Nutrinova Nutrition Specialties
& Food Ingredients GmbH
|
Family ID: |
39863089 |
Appl. No.: |
12/197450 |
Filed: |
August 25, 2008 |
Current U.S.
Class: |
426/548 ;
426/590; 426/592 |
Current CPC
Class: |
A23L 27/31 20160801;
A23L 33/20 20160801; C07K 5/0815 20130101 |
Class at
Publication: |
426/548 ;
426/590; 426/592 |
International
Class: |
A23L 1/227 20060101
A23L001/227; A23L 1/228 20060101 A23L001/228; A23L 2/60 20060101
A23L002/60; C12H 1/00 20060101 C12H001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 8, 2008 |
EP |
08002327.8 |
Claims
1. An oligopeptide according to formula (I) ##STR00003## wherein z
denotes a peptidic condensation product selected from a group of
amino acids comprising: alanine, arginine, asparagine, aspartic
acid, cysteine, glutamic acid, glutamine, histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine or valine; wherein said amino acids
being a .beta.-D- or .beta.-L-amino acid; and wherein for n being
>1 said Zs may be identical or different; n being an integer
from 1 to 5; x being an integer from 1 to 5; and Y being selected
from the group comprising: --H, --OH, --NH.sub.2, --O-Alkyl,
--O--(CO)-Alkyl, --NH-Alkyl, --N(Alkyl).sub.2, --NH--(CO)-Alkyl or
--N((CO)-Alkyl) 2; wherein "Alkyl" being a linear or branched
C.sub.1 to C.sub.5 alkyl rest; and wherein two or more Ys being
identical or different; and said oligopeptide being used as a taste
modulator for one or more comestible compositions.
2. The oligopeptide of claim 1 wherein said amino acids Z being a
D-amino acids, specifically a .beta.-D-amino acids.
3. The oligopeptide use of claim 1 wherein said amino acid Z being
.beta.-D-alanine, being 2, x being 3, and Y being NH.sub.2 or
OH.
4. The oligopeptide of claims 1, wherein said oligopeptide being of
formula (II). ##STR00004##
5. The oligopeptide of claims 1, wherein said oligopeptide being
used as a sweetness enhancer.
6. The oligopeptide of claim 1, wherein said comestible composition
contains at least one natural or artificial sweetener.
7. The oligopeptide of claims 1, wherein said oligopeptide being
water soluble.
8. The oligopeptide claims 1, wherein more than one oligopeptide is
used.
9. The oligopeptide of claims 1, wherein said one or more
oligopeptides being used in an amount between 0.01 mg and 10 g
oligopeptide(s)/kg of the said comestible composition.
10. The oligopeptide of claim 1, wherein said comestible
composition contains mono-, di- or oligosaccharides as
sweeteners.
11. The oligopeptide of claim 1, wherein said comestible
composition contains high fructose corn syrup (HFCS) as a
sweetener.
12. The oligopeptide of claim 1, wherein said comestible
composition being selected from the group comprising: ice cream,
beverages, yoghurts, desserts, spreads, medicinal compositions and
carbohydrated alcoholic and non-alcoholic beverages.
13. A method for the modulation of taste of a comestible
composition comprising the step of adding an oligopeptide according
to formula (I) as defined in claim 1 to said comestible
composition.
14. A method for reducing the concentration of caloric sweeteners
in a comestible composition comprising the step of adding an
oligopeptide according to formula (I) as defined in claim 1 to said
comestible composition.
15. A comestible composition containing an oligopeptide according
to formula (I) as defined in claim 1.
16. The oligopeptide of claim 12 wherein said comestible
composition being a carbonated alcoholic and/or non alcoholic
beverage.
17. The oligopeptide of claim 4, wherein said oligopeptide being
used as a sweetness enhancer.
18. The oligopeptide of claim 4, wherein said comestible
composition contains at least one natural or artificial
sweetener.
19. The oligopeptide of claims 4, wherein said oligopeptide being
water soluble.
20. The oligopeptide of claim 4, wherein more than one oligopeptide
is used.
Description
[0001] The present invention relates to the use of oligopeptides as
taste modulators for comestible compositions containing at least
one sweetener. Furthermore, this invention relates to a method for
the modulation of taste and/or aftertaste of said comestible
compositions as well as to such compositions containing at least
one oligopeptide as taste modulator.
[0002] In this specification, a number of documents are cited, the
entire disclosures of these references (including i. a. scientific
articles, patents and patent applications) are hereby incorporated
herein by reference for the purpose of describing at least in part
the knowledge of those of ordinary skill in the art and for the
purpose of disclosing e.g. compounds, structures (such as T2Rs and
T1Rs mammalian taste receptor proteins) and methods for e.g.
expressing those receptors in cell lines and using the resulting
cell lines for screening compounds with regard to their taste
modulating activity.
[0003] There has been significant recent progress in identifying
useful derivatives of natural flavouring agents, such as for
example sweeteners that are derivatives of natural saccharide
sweeteners, such as for example erythritol, isomalt, lactitol,
mannitol, sorbitol, xylitol. There has also been recent progress in
identifying natural terpenoids, flavonoids, or proteins as
potential sweeteners. See, for example, a recent article entitled
"Noncariogenic Intense Natural Sweeteners" by Kinghorn et al. (Med.
Res Rev (1998)18(5):347-360), which discussed recently discovered
natural materials that are much more intensely sweet than common
natural sweeteners such as sucrose, fructose, glucose, and the
like. Similarly, there has been recent progress in identifying and
commercializing new artificial sweeteners, such as aspartame,
saccharin, acesulfame-K, cyclamate, sucralose, and alitame, etc.;
see an article by Ager et al. (Angew. Chem. Int. Ed. (1998)
37,1802-1817).
[0004] In recent years substantial progress has been made in
biotechnology in general and in better understanding the underlying
biological and biochemical phenomena of taste perception. For
example, taste receptor proteins have been recently identified in
mammals that are involved in taste perception. Particularly, two
different families of G protein coupled receptors believed to be
involved in taste perception, T2Rs and T1Rs, have been identified.
(See, e. g., Nelson et al., Cell (2001) 106(3):381-390; Adler et
al., Cell (2000) 100(6):693-702; Chandrashekar et al., Cell (2000)
100:703-711; Matsunami et al., Number (2000) 404:601-604; Li et
al., Proc Natl Acad Sci USA (2002) 99:4962-4966; Montmayeur et al.,
Nature Neuroscience (2001) 4(S):492-498; U.S. Pat. No. 6,462,148;
and PCT publications WO 02/06254, WO 00/63166 art, WO 02/064631,
and WO 03/001876, and U.S. Patent Publication US 2003-0232407
A1.
[0005] Whereas the T2R family includes over 25 genes that are
involved in bitter taste perception, the T1R family responsible for
sweet perception only includes three members, T1R1, T1R2 and T1R3
(cf. Li et al., Proc. Natl. Acad. Sci. USA (2002) 99 4962-4966).
Recently, it was disclosed in WO 02/064631 and WO 03/001876 that
certain T1R members, when co-expressed in suitable mammalian cell
lines, assemble to form functional taste receptors. It was found
that co-expression of T1R2 and T1R3 in a suitable host cell results
in a functional T1R2/T1R3 "sweet" taste receptor that responds to
different taste stimuli including naturally occurring and
artificial sweeteners (cf. Li et al., cited hereinabove).
[0006] Food, beverages, pleasing products, sweetenings, pet foods,
medicinal products or cosmetics often do have a high content of
sweeteners, which is generally regarded as undesirable in terms of
sweetener related disease development. Here especially diseases
like obesity, diabetes, cardio vascular diseases and others are
prone mainly to high caloric sweeteners. There is good evidence
that increased uptake of high caloric sweeteners, e. g. mono-, di-
and oligosaccharides especially sucrose, is linked to higher levels
of plasma triacylglycerides which is an accepted risk factor for
cardiovascular disease. Likewise increased sugar uptake can be
linked to a physical status which promotes diabetes, obesity or
other diseases. In the food and beverage industry it is state of
the art to replace those troubling sugars like glucose, saccarose,
trehalose and others with fructose.
[0007] The global sweetener market is currently at a scale of 170
million tons per year of sugar-equivalent (units of measurement to
compare amounts of different sweeteners, taking into account their
different sweetness potency) in 2005. This market comprises caloric
sweeteners, high-intensity sweeteners and polyols. The most
important caloric sweetener is refined sugar or sucrose; other
caloric sweeteners are high fructose corn syrup, glucose and
dextrose. High-intensity sweeteners are products that provide the
same sweetness as sugar with less material and therefore fewer
calories. They provide 35 to 10,000 times the sweetness of sugar.
They are also known as low-caloric or dietetic sweeteners or, if
they do not include any calories, non-caloric sweeteners. Apart
from acesulfame-K, other important high-intensity sweeteners are
saccharin, aspartame, cyclamate, stevioside and sucralose. Lastly,
polyols are sugar alcohols, which provide the bulk and texture of
sugar but can be labelled as having fewer calories than sugar.
[0008] For instance the use of high fructose corn syrup (HFCS) as
sweeteners in baked goods (HFCS 90), soft drinks (HFCS 55), sports
drinks (HFCS 42) or in breads, cereals condiments etc. is commonly
accepted. HFCS refers to a group of corn syrups which are
enzymatically processed in order to increase their fructose content
and are then mixed with pure corn syrup (100% glucose) to reach
their final form. The typical types of HFCS are HFCS 90
(approximately 90% fructose and 10% glucose); HFCS 55
(approximately 55% fructose and 45% glucose); and HFCS 42
(approximately 42% fructose and 58% glucose).
[0009] However, conclusions from recent studies can be drawn that
the effects of fructose compared to sucrose on blood glucose,
insulin, leptin, and ghrelin levels exhibit no significant
differences. Taken together there is little or no evidence for the
hypothesis that HFCS is different from sucrose in its effects on
appetite or on metabolic processes involved in fat storage.
[0010] Another strategy to reduce caloric sweeteners, in e. g.
packaged food, is the use of non- or low-caloric artificial
sweeteners like acesulfame-K, saccharin, cyclamate, aspartame,
thaumatin or neohesperidin DC, sucralose, neotame or steriol
glycosides. Here two aspects are of major impact. Firstly these
compounds compared to saccharides have a distinct aftertaste and
secondly there is a permanent discussion whether or not these
sweeteners are carcinogenic.
[0011] It is therefore desirable and an object of the present
invention to find compounds with properties to modulate sweet
taste, or to enhance the sweet taste evoked by a sweetener known in
the art either by being sweet on their own, or being a moderate to
weak sweetener on its own with enhancing attributes for one or more
sweetener(s) known in the art, or most preferably being an enhancer
with no sweetening attributes on its own but the ability to enhance
one or more sweeteners known in the art which are used in
comestible compositions.
[0012] In the art several proposals have been made with regard to
compounds showing taste modulating activity. WO 2006/138512 A2
discloses bis-aromatic amides and their uses as sweet flavour
modifiers, tastants and taste enhancers. U.S. Pat. No. 7,175,872 B2
relates to pyridinium-betain compounds for use as taste
modulators.
[0013] Nevertheless, there remains in the art a need for new and
improved taste modulators of flavouring agents especially for
compounds having no or only very little sweetener potential for the
reasons outlined above.
[0014] The present invention solves these problems.
[0015] One aspect of the invention is the use of an oligopeptide or
of a mixture of different oligopeptides as a taste modulator in
comestible compositions containing one or more natural or
artificial sweeteners examples of which are described above.
Another aspect of the present invention is a method for the
modulation of taste (including aftertaste) of the above mentioned
comestible compositions comprising combining such compositions with
a taste modulating amount of an oligopeptide or mixture of
oligopeptides. And still another aspect of the invention relates to
a comestible composition containing one or more natural or
artificial sweeteners and a taste modulating amount of an
oligopeptide or a mixture of oligopeptides.
[0016] For the purpose of the present invention the following terms
shall have the meanings described below:
[0017] Comestible composition is to be understood in its broadest
sense including but not limited to food, beverages, soft drinks,
pleasing products, sweets, sweetenings, cosmetics such as for
example mouthwash, animal food such as pet foods, and
pharmaceuticals or medicinal products.
[0018] Taste modulator or taste modulation refers to a compound
that modulates the taste (including aftertaste) of a comestible
composition containing one or more natural or artificial
sweeteners. A taste modulator may modulate, enhance potentiate or
induce the taste impression in an animal or a human and preferably
in the sense of enhanced sweet taste.
[0019] Natural and artificial sweeteners are those sweetening
agents known and/or used in the art with respect to comestible
compositions; examples of which are given in the preceding
paragraphs.
[0020] A taste modulating amount refers to an amount of a compound
or compounds capable of modulating the taste of sweetener
containing comestible compositions. The concentration of a taste
modulator needed to modulate or improve the taste of the comestible
composition will of course depend on many variables, including the
specific type of comestible composition and its various other
ingredients, especially the presence of other natural and/or
artificial sweeteners and the concentrations thereof, the natural
genetic variability and individual preferences and health
conditions of various human beings tasting the compositions, and
the subjective effect of the particular compound on the taste of
such sweet compounds.
[0021] Thus, it is not possible to specify an exact "effective
amount". However, an appropriate effective amount can be determined
by one of ordinary skill in the art using only routine
experimentation (see e.g. Ex. 9 of U.S. Pat. No. 7,175,872 and Ex.
53 of WO 2006/138512 A2).
[0022] The oligopeptides which can be used in the present invention
are oligopeptides of the formula (I)
##STR00001##
[0023] wherein [0024] Z denotes the peptidic condensation product
of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic
acid, glutamine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, proline, serine, threonine, tryptophan,
tyrosine or valine wherein any of these amino acids is a .beta.-D-
or .beta.-L-amino acid and wherein for n being >1 the Zs are
identical or different, [0025] n is an integer from 1 to 5 and
[0026] x is an integer from 1 to 5 and [0027] Y denotes --H, --OH,
--NH.sub.2, --O-Alkyl, --O--(CO)-Alkyl, --NH-Alkyl,
--N(Alkyl).sub.2, --NH--(CO)-Alkyl or --N((CO)-Alkyl).sub.2 wherein
"Alkyl" is a linear or branched C.sub.1 to C.sub.5 alkyl rest and
wherein two or more Ys are identical or different.
[0028] All such derivatives can be obtained by methods well known
in the synthesis of oligopeptides.
[0029] Preferred oligopeptides of formula (I) for the use according
to the present invention are those, wherein the amino acids are
D-amino acids, specifically .beta.-D-amino acids.
[0030] Even more preferred is the use of oligopeptides of formula
(I) wherein Z is .beta.-D-alanine, n is 2 and x is 3 and Y is
NH.sub.2 or OH.
[0031] Most preferred is a tripeptide represented by formula
(II)
##STR00002##
[0032] Its generic name is Aladapcin (IUPAC:
2-[2-[(2,6-diamino-6-carbamoyl-hexanoyl)-amino]propanoylamino]propanoic
acid; MW: 331.368 |MF: C13H25N5O5 |XLogP: -4.8. It was first
described by (Shiraishi et al., 1990, J. Antibiot (Tokyo) 43,
634-8); and isolated from bacteria strain Nocardia sp. and is
characterized by the above formula II).
[0033] The preferred taste modulator oligopeptides are water
soluble. Thus, the preferred oligopeptides are those which contain
amino acids which render the resulting oligopeptide water soluble,
such as serine, lysine, or other polar amino acids.
[0034] The oligopeptides according to the invention also include
derivatives. The preferred derivatives of aladapcin are those in
which one or both of the D-alanines in formula I are replaced by
any other D-amino acids such as D-arginine, D-asparagine,
D-aspartic acid, D-cysteine, D-glutamic acid, D-glutamine,
D-histidine, D-isoleucine, D-leucine, D-lysine, D-methionine,
D-phenylalanine, D-proline, D-serine, D-threonine, D-tryptophan,
D-tyrosine, D-valine or glycine or the corresponding L-amino acids.
Thus, Z in formula I above may be any of these amino acids.
[0035] The comestible compositions to which the taste modulating
oligopeptides according to the present invention are added are
preferably compositions containing one or more mono-, di- or
oligosaccharides as sweeteners, and most preferred are compositions
containing high fructose corn syrup or high fructose syrup blends
as sweeteners. Among confectioneries, cereals, ice cream,
beverages, yoghurts, desserts, spreads and bakery products,
nutricosmetics and medicinal compositions, preferably carbohydrated
alcoholic and non-alcoholic beverages like carbonated and
non-carbonated a) soft drinks, b) full calorie soft drinks, c)
sport and energy drinks, d) juice drinks, e) ready-to-drink teas
and other instant soft drinks, are comestible compositions of
special interest for the purpose of the present invention. Most
preferably are those numerous foods in which the liquid sweetener
HFCS, which also constitutes a major source of dietary fructose,
has become a favourite substitute for sucrose e. g. in soft drinks
and many other sweetened beverages as well as in carbonate
beverages, baked goods, canned fruits, jams and jellies, and dairy
products.
[0036] The comestible compositions containing mono-, di- or
oligosaccharides as sweeteners and an oligopeptide according to the
present invention exhibit a taste quality identical or at least
close to the taste of the said saccharides themselves, and
especially a significantly enhanced sweetness.
[0037] The oligopeptides according to the invention and especially
those of the aladapcin type significantly multiply or enhance the
sweetness of known natural and/or artificial sweeteners, even when
used at low concentrations, so that less of the known caloric
sweeteners are required in a comestible composition, while the
perceived taste of the natural sweeteners is maintained or
amplified. This is of very high utility and value in view of the
rapidly increasing incidence of undesirable human weight gain
and/or associated diseases such as diabetes, atherosclerosis,
etc.
[0038] The amount of taste modulator in the inventive comestible
compositions is dependent on the concentration of the natural or
artificial sweeteners contained therein as well as on the presence
of further auxiliary substances such as carbon dioxide, flavors (e.
g. spices, natural extracts or oils), colors, acidulants (e. g.
phosphoric acid and citric acid), preservatives, potassium, sodium
as to mention some of the auxiliaries. The amount desired may
generally be between 0.01 mg and 10 g oligopeptide/kg of the entire
finished comestible composition. The amount is in particular
between 0.01 mg and 1 g oligopeptide/kg, preferably between 0.1 mg
and 500 mg oligopeptide/kg, and especially between 0.1 mg and 50 mg
oligopeptide/kg of the finished comestible composition (=ppm by
weight).
[0039] The oligopeptides of the invention preferably have
sufficient solubility in water and/or polar organic substances, and
mixtures thereof, for formulation at the desired concentration
ranges by simply dissolving them in the appropriate liquids.
Concentration compositions comprising solid but water soluble
substances such as sugars or polysaccharides, and the oligopeptides
described herein can be prepared by dissolving or dispersing the
oligopeptide and soluble carrier in water or polar solvents, then
drying the resulting liquid, via well know processes such as spray
drying.
[0040] The solubility of the oligopeptides of the invention may,
however, be limited in less polar or apolar liquid carriers, such
as oils or fats. In such embodiments it can be desirable to prepare
a very fine dispersion or emulsion of the solid oligopeptide in the
carrier, by grinding, milling or homogenizing a physical mixture of
the oligopeptide and the liquid carrier. The oligopeptides can
therefore in some cases be formulated as sweetener concentrate
compositions comprising dispersions of solid microparticles of the
oligopeptide in the precursor substances. For example, some of the
oligopeptides of the invention can have limited solubility in
non-polar substances such as edible fats or oils, and therefore can
be formulated as sweetener concentrate compositions by milling or
grinding the solid oligopeptide to microparticle size and mixing
with the edible fat or oil, or by homogenizing a dispersion of the
solid oligopeptide and the edible fat or oil, or a comestibly
acceptable analog thereof, such as the Neobee.TM. triglyceride
ester based oils sold by Stephan Corporation of Northfield Ill.
U.S.A.
[0041] It is also possible to prepare solids coated, frosted, or
glazed with the well dispersed compounds of the invention by
dissolving the oligopeptide in water or a polar solvent, then
spraying the solid carrier or composition onto the solid comestible
carrier or substrate.
[0042] By means of the methods described above, many well known and
valuable comestible compositions that currently contain sugar
and/or equivalent saccharide sweeteners can be reformulated to
comprise one or more of the oligopeptides described herein, with a
concomitant ability to reduce the concentration of the sugar and/or
equivalent saccharide sweeteners significantly, e.g. by about 10%
up to as much as 30 to 50%, with a corresponding drop in the
caloric content of the comestible compositions.
[0043] The above described concentrate compositions are then
employed in well known methods to prepare the desired comestible
compositions of the invention.
[0044] Thus, the present invention encompasses different aspects
all belonging to the same inventive concept: [0045] a) the use of
the oligopeptides of the invention as taste modulators for
comestible compositons containing at least one known natural or
artificial sweetener, [0046] b) a method for the modulation of
taste (including aftertaste) of said comestible compositions by
adding one or more oligopeptides of the invention to such
compositions, [0047] c) a method for reducing the concentration of
caloric sweeteners in said comestible compositions by adding one or
more oligopeptides of the invention to said compositions, and
[0048] d) comestible compositions containing at least one known
natural or artificial sweetener and at least one oligopeptide
according to the invention.
EXAMPLES
[0049] Further characteristics of the invention result from the
following examples. In this context single characteristics of this
invention alone or in combination can be realized. The following
examples are provided to illustrate preferred embodiments and are
intended to be illustrative and not limitative of the scope of the
invention.
[0050] Experimental Materials and Methods
[0051] Cell Culture
[0052] Transient transfection/selection of stable HEK293
cells--Transient and stable transfections can be performed with
lipid complexes like calcium phosphate precipitation,
Lipofectamine/PLUS reagent (Invitrogen), Lipofectamine 2000
(Invitrogen) or MIRUS TranslT293 (Mirus Bio Corporation) according
to the manuals. Electroporation can also be a method of choice for
stable transfection of eukaryotic cells.
[0053] The cells are seeded in 6-well plates at a density of
4.times.10.sup.5 cells/well. HEK293 cells are transfected with
linearised plasmids for stable expression of the genes of interest.
After 24 hours, the selection with selecting reagents like zeocin,
hygromycin, neomycin or blasticidin starts. About 50 .mu.l to 300
.mu.l trypsinized transfected cells from a 6-well are seeded in a
100 mm dish and the necessary antibiotic is added in an appropiate
concentration. Cells are cultivated until clones are visible on the
100 mm cell culture plate. These clones are selected for further
cultivation and calcium imaging. It takes about four to eight weeks
to select cell clones which stably express the genes of
interest.
[0054] Calcium Imaging
[0055] Fluo-4 AM assay with stable HEK293 cells--Stable cells are
maintained in DMEM high-glucose medium (Invitrogen) supplemented
with 10% fetal bovine serum (Biochrom) and 4 mM L-glutamine
(Invitrogen). Cells for calcium imaging are maintained in DMEM
low-glucose medium supplemented with 10% FBS and 1.times.
Glutamax-1 (Invitrogen) for 48 hours before seeding. These stable
cells are trypsinized after 48 hours (either with Trypsin-EDTA,
Accutase or TrypLE) and seeded onto poly-D-lysine coated 96-well
assay plates (Corning) at a density of 45,000 cells/well in DMEM
low-glucose medium supplemented with 10% FBS and 1.times.
Glutamax-1.
[0056] After 24 hours, the cells were loaded in 100 .mu.l medium
with additional 100 .mu.l of 4 .mu.M Fluo-4 (calcium sensing dye, 2
.mu.M end concentration; Molecular Probes) in Krebs-HEPES
(KH)-buffer for 1 hour. The loading reagent is then replaced by 80
.mu.l KH-buffer per well. The Krebs-HEPES-buffer (KH-buffer) is a
physiological saline solution including 1.2 mM CaCl.sub.2, 4.2 mM
NaHCO.sub.3 and 10 mM HEPES.
[0057] The dye-loaded stable cells in plates were placed into a
fluorescence microtiter plate reader to monitor fluorescence
(excitation 488 nm, emission 520 nm) change after the addition of
20 .mu.l KH-buffer supplemented with 5x tastants. For each trace,
tastant was added 11.5 seconds after the start of the scan and
mixed two times with the buffer, scanning continued for an
additional 32 seconds, and data were collected every second.
[0058] Data analysis/Data recording--Calcium mobilization was
quantified as the change of peak fluorescence OF) over the baseline
level (F). Data were expressed as the mean S.E. of the )F/F value
of replicated independent samples. The analysis was done with the
software of the microtiter plate reader.
EXAMPLE 1
[0059] Detection of Aladapcin Sweet Enhancer Activity in
Recombinant Human Taste Receptor Dependent T1R2/T1R3 Dependent Cell
Based Assay
[0060] In wild type taste cells--e. g. in the human taste
bud--signal transduction is presumably transduced by the G-proteins
gustducin and/or by G-Proteins of the Galpha-i type. Encountering
sweet ligands the heterodimeric human taste receptor T1R2T1R3
reacts with induction of second messenger molecules; either
induction of the cAMP level in response to most sugars or induction
of the calcium level in response to most artificial sweeteners.
(Margolskee, 2002; J. Biol Chem. 277,1-4)
[0061] To analyze the function and activity of aladapcin the
heterodimeric T1R2/T1R3 sweet taste receptor has been utilized in a
calcium dependent cell based assay. T1R type taste receptors have
been transfected with the multicistronic plasmid vector
pTrix-Eb-R2R3 in a HEK293 cell line stably expressing the
promiscuous mouse G-alpha-15 G-protein.
[0062] For the generation of stable cell lines a multicistronic
expression unit using human taste receptor sequences have been
used. As shown in FIG. 1 the tricistronic expression unit of the
expression vector pTrix-Eb-R2R3 is under the control of the human
elongation factor 1 alpha promoter. Using standard cloning
techniques the cDNA for the receptors ht1R2 and ht1R3 and the cDNA
for the blasticidin S deaminase gene have been cloned. To enable
the translation initiation of each gene of this tricistronic unit
two EMC-virus derived internal ribosomal entry sites (IRES--also
termed Cap-independent translation enhancer (CITE)) have been
inserted. (Jackson et al., Trends Biochem Sci (1990) 15, 477-83;
Jang et al., J Virol (1988) 62, 2636-43.)
[0063] The tricistronic expression unit is terminated by a simian
virus 40 polyadenylation signal sequence. This composition permits
the simultaneous expression of all three genes under the control of
only one promoter. In contrast to monocistronic transcription
units, which integrate independently from each other into different
chromosomal locations during the process of stable cell line
development, the tricistronic transcription unit integrates all
containing genes in one and the same chromosomal locus. Due to the
alignment of the genes, the blasticidin S deaminase gene is only
transcribed in case a full length transcription takes place.
Moreover the polarity of multicistronic transcription units (Moser,
S. et al., Biotechnol Prog (2000) 16, 724-35) leads probably to a
balanced stoichiometry of the receptor genes and their expression
rates in the range of 1:0.7 up to 1:1 for the first two positions
whereas the blasticidin S deaminase gene compared to the receptor
genes in the third position is expressed to a lesser extend.
Assuming that for the functional heterodimeric receptor ht1R2/ht1R3
a 1:1 stoichiometry is needed the lesser polarity effects for the
receptor genes promote the desired stoichiometry whereas the
reduced expression of the deaminase promotes an integration locus
with enhanced transcriptional activity. Generation of stable
T1R2/T1R3 expressing cells have been performed by culturing the
transfected cells in the presence of blasticidine.
[0064] For measurement of human T1R2/T1R3 taste receptor dependent
activity HEK293 cells stably expressing G-alpha-15, human T1R2 and
human T1 R3 were 4.times.10.sup.4 seeded in 96-well plates and
labelled with the calcium sensitive fluorescence dye Fluo4-AM (2
.mu.M) in DMEM culture medium for one hour at 37.degree. C. For the
measurement in a fluorescence plate reader the medium was exchanged
for KH-buffer and incubated for another 15 minutes at 37.degree. C.
Fluorescence measurement of the labelled cells was conducted in a
Flex Station II fluorescence plate reader (Molecular Devices,
Sunnyvale, Calif.). Response to different concentrations of
aladapcin in the presence of 25 mM fructose was recorded as
Fluo4-AM fluorescence increase initiated through the T1R2/T1R3
dependent increase of the second messenger calcium. The applied
fructose concentration was choosen from the results of
preexaminations showing that 25 mM fructose (4.5 g/l) is a
concentration which is barely activating the sweet taste receptors
within this cell based assay (see FIG. 2). Thus a sweetness
enhancing property of a test compound is detectable in the presence
of the sweetener fructose. After obtaining calcium signals for each
sample, calcium mobilization in response to tastants was quantified
as the relative change (peak fluorescence F1-baseline fluorescence
F0 level, denoted as dF) from its own baseline fluorescence level
(denoted as F0). Though rel. RFU is dF/F1. Peak fluorescence
intensity occurred about 20-30 sec after addition of tastants. The
data shown were obtained from at least two independent experiments
and done in triplicates. The fructose enhancing capacity of
aladapcin is depicted in FIG. 2 as primary fluorescence increase
curves and fructose enhancement is given in g/l fructose increase
faciliated by the applied aladapcin concentrations.
LEGENDS
[0065] FIG. 1 shows the multicistronic eucaryotic expression vector
pTrix-Eb-R2R3. The expression of the human taste receptor genes
T1R2, T1R3 and the blasticidin S deaminase (bsd) gene are under the
control of the human elongation factor 1 alpha promoter (P-ef1a).
To confer multicistronic expression on the translational level two
internal ribosomal entry sites (cite-I and cite-II) have been
inserted. The multicistronic unit is terminated by a simian virus
40 polyadenylation site (polyA) and depicted as "cistron" with a
solid black arrow. The prokaryotic origin of replication (ori) and
the kanamycin resistance gene (kan) serve for the propagation,
amplification and selection of the plasmid vector in E. coli.
[0066] FIG. 2 shows the aladapcin activity on sweet taste receptors
(activity as sweet enhancer) in the described cell based assay in
the presence of 25 mM fructose. This is depicted as primary
fluorescence increase (y-axis) over time (sec/x-axis). The
amplification of fructose is dose dependent and the corresponding
hypothetical fructose concentration is indicated in g/l
fructose.
[0067] FIG. 3 shows the results in the same test model without the
presence of fructose which serve as a control measurement. The
results reveal that aladapcin has no sweetener potential, at least
not in the range from 0.1-25 .mu.m.
* * * * *